Enteropathogenic Escherichia coli (EPEC) is a significant cause of profuse watery diarrhea of infants living in developing countries. E. coli serotype O157:H7 is related to EPEC and is of particular concern for public health in the US because this organism causes outbreaks of bloody diarrhea in children and adults due to contaminated meat products, produce and water. A hallmark of EPEC and E. coli O157:H7 infection is their ability to form attaching and effacing (AE) intestinal lesions, causing loss of absorptive tissue, subversion of host cell signaling events and ultimately diarrhea. Genes necessary for the AE phenotype of both EPEC and E. coli O157:H7 are found within a ~35 kb pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE), which encodes a type III secretion system. The LEE-encoded regulator Ler is necessary for elaboration of the type III secretion system and the AE phenotype of these diarrheal pathogens, while the Ler-related H-NS DNA binding protein serves as a silencer of the LEE. [PARAGRAPH] The long-term goal of our research is to determine, on the molecular level, how Ler increases transcription of the genes encoding EPEC type III secretion components. We have previously established that Ler increases transcription of LEE genes, in part, by disrupting H-NS-dependent silencing. However, the precise molecular mechanism of how Ler controls transcription remains to be elucidated. As described in the Progress Report, we have completed an initial characterization of the three functional domains of Ler by producing Ler/H-NS chimeric proteins, assaying for their ability to increase expression of LEE genes and the control operon proU. We have determined that the Ler linker region, connecting the N-terminal oligomerization and C-terminal DNA binding domains, is essential for proper function. Interestingly, placing the Ler linker domain between the N-terminal and C-terminal domains of H-NS creates a protein that increases LEE transcription. Based on these experiments, and helpful comments from reviewers of the initial submission of the competitive renewal, the Research Design and Methods of the grant have been completely rewritten. During this period of support we will use biochemical and biophysical approaches to address two important Specific Aims: 1) Determine the molecular characteristics of Ler that distinguish this protein from H-NS; and 2) Investigate how Ler differentially regulates LEE virulence genes. PUBLIC HEALTH RELEVANCE: With the increasing problem of antibiotic resistant strains, researchers must look for alternative strategies to develop chemotherapeutics against enteropathogenic Escherichia coli (EPEC) and related pathogens. Several studies have indicated that targeting bacterial regulatory pathways may be an effective method of developing such therapeutics. Specifically, we have established that the linker domain of the global regulatory protein Ler- unique in comparison to the linker region of the related H-NS transcriptional silencer- is necessary for proper function of Ler in EPEC. Further elucidation of the molecular mechanisms of Ler action should lead to the development of chemotherapeutic agents directed against EPEC and related E. coli O157 bacteria. [unreadable] [unreadable] [unreadable] [unreadable]